Quantitative differential interference contrast (dic) microscopy and photography based on wavefront sensors

ABSTRACT

A wavefront microscope or camera utilizes a wavefront sensor to measure the local intensity and phase gradient of the wavefront and output image maps based on the intensity and phase gradient. A wavefront sensor provides a metal film having patterned structured two dimensional (2D) apertures that convert a phase gradient of a wavefront into a measurable form onto a photodetector array. A computer is used to analyze the data by separating signals projected and recorded on the array from the different apertures, predict a center of each projection, and sum signals for each projection to display the intensity while determining a center position change/offset from the predicted center to display the phase gradient of the wavefront.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. Section 119(e) ofthe following co-pending and commonly-assigned U.S. provisional patentapplication(s), which is/are incorporated by reference herein:

Provisional Application Ser. No. 61/126,441, filed on May 5, 2008, byXiquan Cui and Changhuei Yang, entitled “QUANTITATIVE DIFFERENTIALINTERFERENCE CONTRAST (DIC) MICROSCOPY BASED ON WAVEFRONT SENSORS,”attorneys' docket number 176.44-US-P1.

This application is related to the following co-pending andcommonly-assigned patent applications, which applications areincorporated by reference herein:

U.S. patent application Ser. No. 11/743,581, filed on May 2, 2007, byXiquan Cui, Xin Heng, Demetri Psaltis, Axel Scherer, and Changhuei Yang,entitled “ON-CHIP PHASE MICROSCOPE/BEAM PROFILER BASED ON DIFFERENTIALINTERFERENCE CONTRAST AND/OR SURFACE PLASMON ASSISTED INTERFERENCE,”attorneys' docket number 176.35-US-U1/CIT-4633, which application claimspriority to the following applications: Provisional Application Ser. No.60/796,997, filed on May 2, 2006, by Xiquan Cui, Xin Heng, and ChanghueiYang, entitled “DIFFERENTIAL INTERFERENCE CONTRAST (DIC) MICROSCOPEBASED ON YOUNG'S INTERFERENCE,” attorneys' docket number 176.35-US-P1(CIT-4633-P); and Provisional Application Ser. No. 60/796,996, filed onMay 2, 2006, by Xin Heng, Xiquan Cui, Axel Scherer, Demetri Psaltis, andChanghuei Yang, entitled “SURFACE PLASMON ASSISTED OPTOFLUIDICMICROSCOPE,” attorneys' docket number 176.35-US-P2 (CIT-4634-P); and

U.S. patent application Ser. No. 11/686,095, filed on Mar. 14, 2007, byChanghuei Yang and Demetri Psaltis, entitled “OPTOFLUIDIC MICROSCOPEDEVICE,” attorneys' docket number CIT-4124-CIP, which application is acontinuation-in-part application of U.S. patent application Ser. No.11/125,718, filed on May 9, 2005, which is a non-provisional of andclaims priority to U.S. provisional patent application Nos. 60/590,768,filed on Jul. 23, 2004, and 60/577,433, filed on Jun. 4, 2004.Application Ser. No. 11/686,095 is also a non-provisional of, and claimsthe benefit of the filing date of U.S. provisional patent applicationNo. 60/783,920, filed on Mar. 20, 2006.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to microscopy and photography,and in particular, to differential interference contrast (DIC)microscopy and photography based on wavefront sensors.

2. Description of the Related Art

(Note: This application references a number of different publications asindicated throughout the specification by reference numbers enclosed inbrackets, e.g., [x]. A list of these different publications orderedaccording to these reference numbers can be found below in the sectionentitled “References.” Each of these publications is incorporated byreference herein.)

Differential interference contrast (DIC) microscopy renders excellentcontrast for optically transparent biological samples without the needto introduce any exogenous contrast agents into the samples. Due to thisnoninvasive nature, DIC microscopes are widely used in biologylaboratories. However, conventional DIC techniques have severallimitations. One major disadvantage is that the conventional DICmicroscope translates phase variations into amplitude (intensity)variations, and therefore phase variations cannot be easily disentangledfrom amplitude variations that arise from sample absorption and/orscattering [1]. In other words, conventional DIC microscopes areinherently qualitative as a consequence of nonlinear phase gradientresponse and entanglement of amplitude and phase information. Inaddition, conventional DIC images of birefringent samples can havesignificant artifacts as the conventional DIC microscope depends onpolarized light and uses polarization in its phase imaging strategy.

SUMMARY OF THE INVENTION

One or more embodiments of the invention provide a quantitative DICmicroscopy or photography method based on a wavefront sensor. Such asensor may be either a structured-aperture (SA) wavefront sensor or amicrolenses aperture based sensor (i.e., a Shack-Harmann sensor).Benefiting from the unique features of such a wavefront sensor, a DICmicroscopy or photography system separates the amplitude and the phasegradient information of the image wavefront, and forms quantitativeintensity and DIC images of the sample with good resolution (˜2 μm orless). Furthermore, since embodiments of the invention utilizeunpolarized light and contain no polarization-dependent components,birefringent samples can be imaged (e.g., potato starch storagegranules) without artifacts. Finally, unlike most of the recentlydeveloped quantitative phase microscopy techniques, embodiments of theinvention can be used with a standard microscope light-source [11][12].

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings in which like reference numbers representcorresponding parts throughout:

FIG. 1 illustrates a conventional DIC microscope system that operates byinterfering slightly displaced duplicate image light fields;

FIG. 2 illustrates a structured aperture differential contrastmicroscope that operates by interfering light from two adjacent pointson the image light field in accordance with one or more embodiments ofthe invention;

FIG. 3 illustrates a configuration for a DIC microscopy based wavefrontsystem using a single pixel wavefront sensor scheme in accordance withone or more embodiments of the invention;

FIG. 4 illustrates a configuration for a DIC microscopy based wavefrontsystem using a wavefront sensor array scheme in accordance with one ormore embodiments of the invention;

FIG. 5 illustrates a configuration of a wavefront sensor used inaccordance with one or more embodiments of the invention;

FIG. 6( a) illustrates a configuration of a structured aperturewavefront sensor in accordance with one or more embodiments of theinvention;

FIG. 6( b), 6(d), and 6(f) illustrate different patterned structuredapertures used in the configuration of FIG. 6( a) while FIGS. 6( c),6(e), and 6(g) illustrate resulting images from the patterned structuredapertures of FIGS. 6( b), 6(d), and 6(f) respectively;

FIG. 7 illustrates an example of a side-view of a DIC microscope systemconstructed by an SAI wavefront sensor array in accordance with one ormore embodiments of the invention;

FIG. 8 illustrates a Shack-Hartmann wavefront sensor in a DIC microscopesystem in accordance with one or more embodiments of the invention;

FIG. 9 is a flowchart illustrating the processing of data received in aphotodetector array in accordance with one or more embodiments of theinvention;

FIG. 10A illustrates a side view of a wavefront sensor in accordancewith one or more embodiments of the invention;

FIG. 10B illustrates a top down view of a photodetector array of FIG.10A with signals received thereon in accordance with one or moreembodiments of the invention;

FIG. 11 is an exemplary hardware and software environment used toimplement one or more embodiments of the invention;

FIG. 12( a) illustrates a configuration of a structured aperturedifferential contrast microscope used in accordance with one or moreembodiments of the invention;

FIG. 12( b) illustrates a graph of an offset of a zero-orderinterference spot of the structured aperture of FIG. 12( c) andwavefront gradient in accordance with one or more embodiments of theinvention;

FIG. 12( c) illustrates a scanning electron microscope image of astructured aperture defined on silver film in accordance with one ormore embodiments of the invention;

FIG. 12( d) illustrates a resulting interference pattern of thestructured aperture recorded by a CMOS (complementarymetal-oxide-semiconductor) image sensor in accordance with one or moreembodiments of the invention;

FIGS. 13( a)-13(e) illustrates images of starfish embryo. FIG. 13( a)illsutrates a conventional transmission microscope image; FIG. 13( b)illustrates a conventional DIC microscope image; FIG. 13( c) illustratesintensity, FIG. 13( d) illustrates X DIC phase images from a structuredaperture DIC microscope, and FIG. 13( e) illustrates Y DIC phase imagesfrom a structured aperture DIC microscope;

FIGS. 14( a)-(e) illustrates images of potato starch storage granules inmicroscope immersion oil; FIG. 14( a) illustrates a conventionalmicroscope image; FIG. 14( b) illustrates maltese-cross-like patternartifacts in a conventional DIC image; FIG. 14( c) illustrates intensityand artifact free; FIG. 14( d) illustrates X DIC phase images from astructured aperture DIC microscope, and FIG. 14( e) illustrates Y DICphase images from a structured aperture DIC microscope;

FIG. 15 illustrates a completed wavefront-sensing sensor chip (topleft), a camera port housing for the chip (top right), and the use ofthe entire unit plugged into a camera port of a microscope in accordancewith one or more embodiments of the invention;

FIG. 16 illustrates the approach for the propagation of light inaccordance with one or more embodiments of the invention; and

FIG. 17 illustrates an approach for the propagation of light using acomputer (17(a)) and using a microscope (17(b) and 17(c)) in accordancewith one or more embodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the following description, reference is made to the accompanyingdrawings which form a part hereof, and which is shown, by way ofillustration, several embodiments of the present invention. It isunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the present invention.

Overview

One or more embodiments of the invention provide a mechanism to create asimple, quantitative and birefringent-artifact-free DIC microscopy orphotography method with wavefront sensors.

Configuration of the Quantitative Differential Interference Contrast(DIC) Microscopy Based on Wavefront Sensors

Using conventional microscopy, a sample may be illuminated and aphotodetector may be used to receive the defracted light. Two types ofinformation are desirable based on the illumination—(1) thenumber/distribution of photons; and (2) the direction/wavefrontslope/phase gradient of the photons. Conventional microscopy fails toprovide the phase gradient of the photons defracted by a sample.Further, as described above, due to the dependency on polarized light,conventional techniques further fail to provide accurate results withbirefringement samples (i.e., samples that have double refraction wherethe direction of light defracted through the sample will depend on thepolarization of the light). Accordingly, what is needed is a mechanismthat is not dependent on polarized light that is further able to providethe phase gradient of the illuminating light and sample.

Embodiments of the invention provide the ability to measure both (1) thenumber/distribution of photons, and (2) the phase gradient of thephotons on birefringent and homogeneous samples. A DIC microscopy-basedsystem of embodiments of the invention may utilize wavefront sensors intwo different configurations: (1) a single pixel wavefront sensorscheme; or (2) a wavefront sensor array scheme. To better understandthese two configurations, a close examination of convention DICmicroscopy is useful.

In essence, a conventional DIC microscope operates by first creating twoidentical illumination light fields exploiting polarization selection.FIG. 1 illustrates a conventional DIC microscope system that operates byinterfering slightly displaced duplicate image light fields. Asillustrated, the light fields 100 are laterally displaced(displacement=a) with respect to each other (along the x-direction) andare transmitted through the sample 102. A net phase lag 104 (typicallyπ/2) is then introduced on one of the transmitted image light fields.The two light fields 100 are allowed to interfere with each other at theimage plane 106. More simply, the process is equivalent to simplyduplicating the transmitted image light field, laterally displacing thecopy slightly (i.e., at 104) and interfering the two light fields (atimage plane 106).

Mathematically, this implies that the observed conventional DICintensity image from a microscope with a magnification factor of M isgiven by:

$\begin{matrix}{{I_{DIC}\left( {x,y} \right)} = {{\psi_{DIC}\left( {x,y} \right)}}^{2}} \\{= {{B\left( {x,y} \right)} + {{C\left( {x,y} \right)}*{\sin\left( {{\arg \left( {\psi \left( {{x - {\Delta/2}},y} \right)} \right)} -} \right.}}}} \\\left. {\arg \left( {\psi \left( {{x + {\Delta/2}},y} \right)} \right)} \right) \\{\approx {{B\left( {x,y} \right)} + {{C\left( {x,y} \right)}*\left( {{\arg \left( {\psi \left( {{x - {\Delta/2}},y} \right)} \right)} -} \right.}}} \\\left. {\arg \left( {\psi \left( {{x + {\Delta/2}},y} \right)} \right)} \right)\end{matrix}$

where B(x,y)=|(ψ(x−Δ/2,y)|²+|(ψ(x+Δ/2,y)|²,C(x,y)=2|(ψ(x−Δ/2,y)∥(ψ(x+Δ/2,y)|, and ψ(x,y) is the image wavefront asrelayed by the microscope for each light field, ψ_(DIC)(x,y) is the DICimage wavefront, and Δ=Ma is the relative displacement of the imagesassociated with the light fields. The last expression in Eq. (1) isvalid only in situations where the phase difference is small.

A similar phase comparison can also be performed by acquiring a simplemicroscope image of the object and selectively combining-and-interferingthe light fields at two adjacent points of the image (separation=a′) asillustrated in FIG. 2. Such a phase comparison is employed in a DICimaging method of one or more embodiments of the invention.

Single Pixel Wavefront Sensor Scheme

A first configuration for a DIC microscopy-based wavefront system isthat of a single pixel wavefront sensor scheme as illustrated in FIG. 3.The light source 300 illuminates a sample 302 which defracts/refractsthe light into a wavefront shape 304.

A wavefront relay system 308 is used to project the wavefront 304generated by the sample 302 onto a single pixel wavefront sensor 310.The sensor 310 measures the local intensity (i.e., number/distributionof the photons) and phase gradient of the wavefront 304 induced by thesample 302 which conjugates with the sensor pixel 310. Byraster-scanning 306 the sample 302 or the sensor pixel 310,two-dimensional (2D) maps of the local intensity/distribution and phasegradient of the wavefront 304 modulated by the sample 302 can beachieved at the same time.

Wavefront Sensor Array Scheme

A second configuration for a DIC microscopy based wavefront system isthat of a wavefront sensor array scheme as illustrated in FIG. 4.Similar to FIG. 3, the light source 400 illuminates the sample 402 whichdefracts/refracts the light into a wavefront shape 404.

The relay system 408 is used to project the wavefront 404 generated bythe sample 402 onto a wavefront sensor array 410. Each pixel of thewavefront sensor array 410 measures the local intensity/distribution andphase gradient of the wavefront 404 induced by the sample point 402which conjugates with the sensor pixel. In this case, 2D maps of thelocal intensity/distribution and phase gradient of the wavefront 404modulated by the sample 402 are obtained naturally at the same time. Inaddition, the sample 402 or the sensor array 410 can be raster-scannedto form more densely sampled images.

Configuration Benefits and Alternatives

Since embodiments of the invention do not depend on the polarization ofthe illumination, artifact-free DIC images can be obtained (usingunpolarized illumination), for both birefringent and homogenous samples.In addition, the imaging scan 306 may be performed in many other ways.For example, a 2D raster scan 306 can be replaced by a one-dimensional(1D) OFM (opto-fluidic microscope) scanning scheme as described in U.S.patent application Ser. Nos. 11/686,095 and 11/743,581 which areincorporated by reference herein. Further, sample scanning can bereplaced by wavefront sensor 310/410 scanning. Lastly, embodiments ofthe invention can be used to target transparent samples, cell level orlarger. In this regard, there is no constraint with respect to the sizeof the sample.

In view of the above described configurations, embodiments of theinvention provide a new DIC microscopy method where the DICfunctionality of a microscope is integrated onto the detector. Suchintegration is advantageous over convention DIC microscopy methods whichuse bulky optical elements in the microscope. Accordingly, embodimentsof the present invention are more compact, less expensive, less tediousin training and preparation, and more convenient to operate by usersthan conventional DIC methods.

Use in a microscope can involve a wavefront-sensing sensor chip that isplaced into a camera-port compatible housing for the chip. The entireunit can be plugged into the camera port of a microscope and used tocapture phase images.

Nonetheless, embodiments of the invention can be utilized in adaptiveoptics as part of a wavefront sensing component. Adaptive opticsoperates by measuring distortions in a wavefront and compensating forthem with a spatial phase modulator such as a deformable mirror orliquid crystal array.

Wavefront Sensors

Multiple different types of wavefront sensors 310/410 may be used inaccordance with one or more embodiments of the invention. The wavefrontsensor, which can measure the local intensity/distribution and phasegradient of the wavefront modulated by the sample at the same time, canbe implemented in several ways. Two different cases are presented: (1)structured aperture interference (SAI) and (2) Shack-Hartmann schemes.Both sensors can be used as either a single pixel 310 or an arrayedwavefront sensor 410, and form quantitative DIC microscopes of theconfigurations described above.

Structured Aperture Interference (SAI) Sensor

The basic concept of the SAI sensor is similar to the Young'sdouble-slit phase decoding scheme described in U.S. patent applicationSer. Nos. 11/743,581 and 11/686,095, which are fully incorporated byreference herein.

FIG. 5 illustrates a configuration of a wavefront sensor 310/410 used inaccordance with one or more embodiments of the invention. A light source500 is used to illuminate a sample/specimen 502. A metal film 504 hasapertures 506 (i.e., 506A and 506B) which are used to focus thedefracted/refracted light from the sample (i.e., in accordance withYoung's interference 508) onto a photodetector array such as chargecoupled device (CCD) 510. The photodetector array 510 can be used todetermine the light intensity distribution 512. The local phase gradientof the wavefront 304/404 (that is focused via apertures 506) isdetermined by examining the offset of the zero diffraction order on theback photodetector array 510. The local intensity 512 of the wavefrontis determined by integrating the diffraction signal on the backphotodetector array 510.

One or more embodiments of the invention utilize varieties of 2Dstructured apertures including four holes, pedal-shaped, a single hole,a ring or Fresnel zone plate (some of them are illustrated in FIG. 6).As illustrated in FIG. 6( a), the incident light field 500 (e.g.,received from the wavefront relay system 308/408) passes throughapertures in the metal (e.g., silver) film where the light is projected(e.g., through a viscous polymer such as SU-8 resin) onto an imagesensor (e.g., CMOS image sensor). The light may be projected atdifferent angles (e.g., left side of FIG. 6( a) v. right side of FIG. 6(a) resulting in a different projection onto the image sensor 510.

FIGS. 6( b), 6(d), and 6(f) illustrate the different structured aperturepatterns (via scanning electron microscope images) while FIGS. 6( c),6(e), and 6(g) illustrate the resulting interference pattern of therespective aperture patterns recorded by the photodetector/image sensor510. FIG. 6( b) and 6(c) illustrate a pedal-shaped aperture andresulting interference patter; FIG. 6( d) and 6(e) illustrate a four (4)structured aperture and resulting interference pattern; and FIGS. 6( f)and 6(g) illustrate a 4-hole structured aperture (with round holes) andresulting interference pattern. FIG. 6( h) illustrates a Fresnel-zoneplate structured aperture with a circular frame. Using a Fresnel zoneplate or ring based structured aperture, one can shrink a projectedsport FWHM (full width half maximum) by appropriate design.

With these 2D structured apertures (i.e., illustrated in FIGS. 6( b),6(d), and 6(f) as examples), one can measure the local phase gradient ofthe wavefront 304/404 along both orthogonal directions X and Y at thesame time. As used herein, such an aperture-based phase decoding schemeis referred to as SAI wavefront sensing.

As illustrated in FIG. 6, the sensor of the invention 310/410 includesthe metal film based apertures 504 and phototector/image sensor 410. Thesensor 510 is used to receive the sample refracted incident light field.Wherever the projection is received onto the sensor 510, the resultingimage may be used to represent a single pixel. As in FIG. 3, rasterscanning may be used to capture an image of the entire sample.Alternatively, as in FIG. 4, multiple sensors 310/410 may be arranged ina 2D array to capture the entire sample in a single snapshot.

The 4-hole aperture and single hole aperture sensor measures the spatialphase gradient of the light field. Mathematically, one measures:

G _(x)(x,y)=k _(x)(x,y)/k _(o)≈(dφ(x,y)/dx)/k _(o)

G _(y)(x,y)=k _(y)(x,y)/k _(o)≈(dφ(x,y)/dy)/k _(o)

A(x,y)

with an aperture sensor.

As such, one can mathematically reconstruct the observed wavefront atthe sensor by combining the information appropriately. One approach is:

ψ_(measured)(x, y, z) = A_(measured)(x, y, z)exp ( k_(o)(∫₀^(x)G_(x)(x, y = 0)x + ∫₀^(y)G_(y)(x, y)y))

Numerous approaches for reconstructing a field distribution exist(unwrapping). The methods should all return the same answer if the SNR(signal to noise ratio) of the measurement approaches infinity. Themethods vary in their performance based on the quantity and type ofnoise present in the measurements.

FIG. 7 illustrates an example of a side-view of a DIC microscope system(and wavefront camera system as described below) constructed by an SAIwavefront sensor array in accordance with one or more embodiments of theinvention. The light source 700 illuminates 700A the sample 702 causinga wavefront 704 to pass through a relay system such as lens 706. Thewavefront passes through SAI array 708 which causes the light to projectonto photodetector array 710. As illustrated, FIG. 7 depicts a3-aperture SAI array 708.

Shack-Hartmann Wavefront Sensor

One or more embodiments of the invention utilize a Shack-Hartmannwavefront sensor in a DIC microscope system (and wavefront camera systemdescribed below) as illustrated in FIG. 8. As with the SAI wavefrontsensor implementation, a light source 800 illuminates 800A a sample 802to generate a wavefront 804 which passes through a wavefront relaysystem such as lens 806. The Shack-Hartmann sensor consists of an array808 of microlenses of the same focal length. Each microlens in the array808 focuses the local wavefront 804 across each lens and forms a spotonto a photonsensor array 810. The local phase gradient of the wavefront804 can then be calculated from the position of the focal spot on thesensor 810. The history and principles of Shack-Hartmann wavefrontsensing is further described in [3] and [4]. However, prior artShack-Hartmann sensors have been limited in their use and have not beenused in microscopy or DIC/phase based microscopy.

It is also worth noting that the diffraction spot size in SAI wavefrontsensors and the focal spot size in the Shack-Hartmann wavefront sensorscan be used to determine the spread of wave vector k at any given imagepoint. It is useful for rendering images where the extent of scatteringis plotted (amongst other things).

Wavefront Detection

SAI wavefront sensors can be used in a variety of wavefront sensingapplications including adaptive optics [3], optical test [3], adaptivemicroscopy [5], retina imaging [6], etc. In an SAI wavefront senor ofthe invention, structured apertures are used on a metal film to convertthe phase gradient of a wavefront into a measurable form—the offset ofthe projection of the aperture. Shack-Hartmann wavefront sensors utilizea microlenses array to perform the conversion. The simple structuredapertures provides the ability to build the wavefront sensor in aneasier and more cost-effective way than Shack-Hartmann sensors. Inaddition, the spacing between structured apertures can be much shorterthan the one between the microlenses of a Shack-Hartmann sensor whichare usually greater than 100 μm and provide wavefront sensing targetsthat are slowly invariant wavefronts. Further, the SAI wavefront sensorprovides for high spatial resolution and much denser wavefront samplingfor use in detecting complicated wavefronts generated by many biologicalsamples.

One may note that wavefront sensing using SAI and Shack-Hartmannwavefront sensors does not only apply to monochromatic illumination, butalso applies to broad band light illumination. The feasibility of suchbroad band light illumination is described in [13] which is fullyincorporated by reference herein. In this regard, wavefront sensingapplies to a monochromatic light field distribution in which k is welldefined at each point on the image plane. However, wavefront sensing mayalso be used for a broadband light source and with situations where k atany given point may be a mix of different wave vectors. In this regard,wavefront sensors can be used with broadband light illumination,monochromatic illumination with mixed k, and broadband lightillumination with mixed k. Such capabilities may be better understoodwith a description of computed depth sectioning ability.

For purposes of the following discussion, assume that a light field islinearly polarized and that no interaction of the light field willdepoloarize or in any other way disrupt polarization. Under a firstconcept, the light field at any given plane can be fully described by acomplete set of spatially varying amplitude and phase information. Inother words, a light field at plane z can be described by:

ψ(x,y,z)=A(x,y,z)exp(iφ(x,y,z))

Under a second concept, Huygen's Principle states that the light fieldat a earlier or later (higher or lower z value) can be calculated fromthe light field at plane z. In other words, a known function (f)connects:

ψ(x,y,z+Δz)=ƒ(ψ(x,y,z),Δz)

The function f is well known and studied in EM (electromagnetic) theory(see [14]). This computation assumes that the absence of unknownscattering objects between plane z and plane (z+Δz).

These two concepts are powerful when applied to phase imaging in thecontext of embodiments of the present invention. It implies that if wecan measure the phase and amplitude distribution at the plane of thesensor (optofluidic microscope [OFM] floor—see details describedherein), one can calculate and render the light field distributions atdifferent heights above the sensor. These light field distributions are,in effect, images at those chosen planes.

FIG. 16 illustrates the approach for the propagation of light inaccordance with one or more embodiments of the invention. Thepropagation of the light field is governed by Maxwell's equationsentirely. If one measures the phase and amplitude distribution of thelight field 1600 at the sensor 1602, such information can be used tocalculate the light field distribution at any given plane above thesensor (or below the sensor). The amplitude distribution of the computedlight field is equivalent to the traditional microscope image taken atthe focal plane 1604 set at z+z. This treatment is strictly true if nounknown object (e.g., sample) is present between the plane z (i.e., atthe sensor 1602) and z+z (1604).

FIG. 17 illustrates an approach for the propagation of light using acomputer (17(a)) and using a microscope (17(b) and 17(c)). The imagingof an unknown but weak scatterer can be performed computationally (e.g.,as illustrated in FIG. 17( a)) by using the same mathematical frame workas described above—by ignoring the presence of the scatterer during thecalculation and back-computing the light field at z+Δz.

The amplitude distribution of the computed light field is equivalent tothe traditional microscope image (e.g., as illustrated in FIG. 17( b))taken at the focal plane set at z+Δz. In fact, the amplitudedistribution is identical. In the traditional microscope (i.e., FIGS.17( a) and 17(b)), the calculation is performed optically by the opticalelements. By adjusting the optics, you can bring different planes intofocus but effectively, one is making slight adjustments to the opticalcomputing process. As an example, FIG. 17( a) illustrates a microscopebased approach where the optics are tuned to focus on the plane z+Δz1702. The resulting distribution of such a focal plane 1702 is apparentat the sensor 1704. Similarly, in FIG. 17( c), the optics are tuned tofocus on plane z+Δz′ 1706 which is also apparent at sensor 1704.

The process may not be perfect because one may fail to achieve a goodimage if the scatterer is thick and/or highly scattering because theassumption that the scatterer is ignorable in the computation process isviolated in this case. However, this problem affects computation-baseddepth sectioning and optical-based sectioning equally.

The axiom of ‘no free lunch’ applies equally in both situations. Forthin tissue sections or cell samples, the distortion is nominallytolerable. In practical situations, one can typically deal with a 100microns thick tissue sample before distortion starts becomingsignificant.

One may also note that with computation based sectioning, a sensorcapable of spatially measuring amplitude and phase may be needed.Further, the signal to noise ratio (SNR) of the sensor movements mayhave to be high. Otherwise, the computed images may be poor in quality.

Wavefront Camera

Embodiments of the invention can also be implemented by insertion of aSAI wavefront sensor (FIG. 7) or a Shack-Hartmann wavefront sensor (FIG.8) into a camera system instead of microscope. Such an embodiment incamera imaging provides similar advantages and benefits to that of amicroscopy based embodiment. For example, one can not only capture theintensity information of the projected object wavefront onto the imagesensor, but the phase gradient of the projected object wavefront canalso be detected. In FIG. 7 the light source 700 illuminates 700B anobject 702 wherein the remaining elements are similar to the microscopybased device described above. Similarly, in FIG. 8, the light source 800illuminates 800B the object 802 (which could be a person, or otherobject that is being captured via the wavefront camera) with theremaining elements similar to the microscopy based device describedabove. As illustrated in FIGS. 7 and 8, the illumination 700B and 800Breflects off of the object 802 to generate a wavefront 704/804 that ispassed to the lens and eventually reaches the photodetector array710/810.

Data Analysis

Once a wavefront has been received in the photodetector array,embodiments of the invention provide the ability to analyze the receiveddata and output an image or information based on such data analysis.FIG. 9 is a flowchart illustrating the processing of the data receivedin the photodetector array in accordance with one or more embodiments ofthe invention. FIG. 9 will be described in relation to FIG. 10A whichillustrates a side view of a wavefront sensor and FIG. 10B whichillustrates a top down view of the photodetector array 1010 with signalsreceived thereon.

In FIG. 10A, a wavefront 1004 (e.g., from a wavefront relay system) isreceived at the SAI array 1008. The SAI array 1008 causes the wavefront1004 to project onto the photodetector array 1010. Thedistribution/intensity of the wavefront signals 1004 received on thephotodetector array 1010 is illustrated in FIG. 10A as wave 1012 and inFIG. 10B by the circles 1012. In this regard, at step 900, a snapshot(via the illumination via wavefront 1004) is taken at the photosensorarray 1010. At step 900, the projection of every hole is also recorded.It may be noted that while there are multiple photosensors in thephotodetector array 1010, the different photosensors may be cumulativelyused or a single photosensor may be used to provide a single datapointthat is used in accordance with embodiments of the invention.

At step 902, the projection of the hole/aperture is separated from theother projections. Such a separation may be performed by suppressing thecrosstalk from neighbor hole projections iteratively. Any variety ofmethods may be used to separate the projections of the wavefront 1004through the SAI array 1008. For example, in one embodiment, a maximumintensity value (e.g., in FIG. 10A) is found and a determination is maderegarding when the intensity value decreases. The wave projection isfollowed to determine when the intensity value begins to rise again(i.e., signaling the wavefront from an adjacent aperture). The midpointbetween two maximum intensity values can be used to suppress cross talkand separate the projections. Alternatively, the height 1014 (i.e.,between the SAI array 1008 and the photodetector array 1010) can beestablished and the distribution 1012 can be examined to determine thepeak/maximum pixel. A defined number of pixels can be used on each sideof the peak signal (e.g., from the center of the projection) to definethe different aperture based projections. However, any variety oftechniques can be used to separate out the pixels/projections. Such aseparation is useful to map the different regions in the photodetectorarray 1010 to the appropriate aperture in the SAI array 1008.

At step 904, the center of the projection of the hole/aperture ispredicted. Any methodology can be used for this prediction including theexamination of the intensity values (e.g., as described above withrespect to the separation of the projection of the holes). Step 904 mayalso include the summing of all signals of the hole projections.

Steps 900-904 are performed twice. The first time steps 900-904 areperformed, no sample is used. Such a performance serves to initializethe system and determine base measurements to be used to determine thephase gradient. The second time steps 900-904 are performed, a sample isutilized such that the projections will be defracted differently(compared to the projections without the sample).

At step 906, the center position change of the hole projection (i.e.,the offset 1016) is determined. Such an offset may be determined in boththe X and Y directions. This center position change/offset 1016 isdirectly related to the phase gradient of the wavefront at the hole(i.e., the offset divided by the height may provide the phase gradient).In addition, at step 906, the total signal of the hole projection isdetermined which is proportional to the intensity of the wavefront atthe hole.

The phase gradient of the wavefront along any direction can becalculated based on the phase gradient of the wavefront along

${X\left( \frac{\partial\varphi}{\partial x} \right)}\mspace{14mu} {and}\mspace{14mu} {Y\left( \frac{\partial\varphi}{\partial y} \right)}$

directions acquired as described above. It is because the phase of awavefront is a fixed scalar potential function, and the phase gradientof the wavefront along any direction

$\frac{\partial\varphi}{\partial\overset{\rightharpoonup}{n}}$

can be represented as the inner product of the unit direction vector{right arrow over (n)} and the gradient vector of the phase {right arrowover (∇)}φ.

${\frac{\partial\varphi}{\partial\overset{\rightharpoonup}{n}}} = {{\overset{\rightharpoonup}{n} \cdot {\overset{\rightharpoonup}{\nabla}\varphi}} = {\overset{\rightharpoonup}{n} \cdot \left( {{\frac{\partial\varphi}{\partial x}\overset{\rightharpoonup}{i}} + {\frac{\partial\varphi}{\partial y}\overset{\rightharpoonup}{j}}} \right)}}$

At step 906, the data can be output. For example, the angles of theprojections (i.e., the direction of the light at a particular point) canbe plot into a 2D image that reflects how the light is detracted by thesample. The structure of the sample can therefore be displayed in animage using the phase gradient.

Such processing of FIG. 9 may be performed by the computer illustratedin FIG. 11. In this regard, FIG. 11 is an exemplary hardware andsoftware environment 1100 used to implement one or more embodiments ofthe invention. Computer 1100 may be a user computer, server computer, ormay be a database computer. The computer 1102 comprises a generalpurpose hardware processor 1104A and/or a special purpose hardwareprocessor 1104B (hereinafter alternatively collectively referred to asprocessor 1104) and a memory 1106, such as random access memory (RAM).The computer 1102 may be coupled to other devices, includinginput/output (I/O) devices such as a keyboard 1114, a mouse device 1116and a printer 1128.

In one embodiment, the computer 1102 operates by the general purposeprocessor 1104A performing instructions defined by the computer program1110 under control of an operating system 1108. The computer program1110 and/or the operating system 1108 may be stored in the memory 1106and may interface with the user and/or other devices to accept input andcommands and, based on such input and commands and the instructionsdefined by the computer program 1110 and operating system 1108 toprovide output and results.

Output/results may be presented on the display 1122 or provided toanother device for presentation or further processing or action. In oneembodiment, the display 1122 comprises a liquid crystal display (LCD)having a plurality of separately addressable liquid crystals. Eachliquid crystal of the display 1122 changes to an opaque or translucentstate to form a part of the image on the display in response to the dataor information generated by the processor 1104 from the application ofthe instructions of the computer program 1110 and/or operating system208 to the input and commands. The image may be provided through agraphical user interface (GUI) module 1118A. Although the GUI module1118A is depicted as a separate module, the instructions performing theGUI functions can be resident or distributed in the operating system1108, the computer program 1110, or implemented with special purposememory and processors.

Some or all of the operations performed by the computer 1102 accordingto the computer program 1110 instructions may be implemented in aspecial purpose processor 1104B. In this embodiment, the some or all ofthe computer program 1110 instructions may be implemented via firmwareinstructions stored in a read only memory (ROM), a programmable readonly memory (PROM) or flash memory within the special purpose processor1104B or in memory 1106. The special purpose processor 1104B may also behardwired through circuit design to perform some or all of theoperations to implement the present invention. Further, the specialpurpose processor 1104B may be a hybrid processor, which includesdedicated circuitry for performing a subset of functions, and othercircuits for performing more general functions such as responding tocomputer program instructions. In one embodiment, the special purposeprocessor is an application specific integrated circuit (ASIC).

The computer 1102 may also implement a compiler 1112 which allows anapplication program 1110 written in a programming language such asCOBOL, Pascal, C++, FORTRAN, or other language to be translated intoprocessor 1104 readable code. After completion, the application orcomputer program 1110 accesses and manipulates data accepted from I/Odevices and stored in the memory 1106 of the computer 1102 using therelationships and logic that was generated using the compiler 1112.

The computer 1102 also optionally comprises an external communicationdevice such as a modem, satellite link, Ethernet card, or other devicefor accepting input from and providing output to other computers or fromthe photodetector array device of the invention.

In one embodiment, instructions implementing the operating system 1108,the computer program 1110, and the compiler 1112 are tangibly embodiedin a computer-readable medium, e.g., data storage device 1120, whichcould include one or more fixed or removable data storage devices, suchas a zip drive, floppy disc drive 1124, hard drive, CD-ROM drive, tapedrive, etc. Further, the operating system 1108 and the computer program1110 are comprised of computer program instructions which, whenaccessed, read and executed by the computer 1102, causes the computer1102 to perform the steps necessary to implement and/or use the presentinvention or to load the program of instructions into a memory, thuscreating a special purpose data structure causing the computer tooperate as a specially programmed computer executing the method stepsdescribed herein. Computer program 1110 and/or operating instructionsmay also be tangibly embodied in memory 1106 and/or data communicationsdevices 1130, thereby making a computer program product or article ofmanufacture according to the invention. As such, the terms “article ofmanufacture,” “program storage device” and “computer program product” asused herein are intended to encompass a computer program accessible fromany computer readable device or media.

Of course, those skilled in the art will recognize that any combinationof the above components, or any number of different components,peripherals, and other devices, may be used with the computer 1102.

Although the term “user computer” is referred to herein, it isunderstood that a user computer 1102 may include portable devices suchas cellphones, notebook computers, pocket computers, or any other devicewith suitable processing, communication, and input/output capability.

Implementation Details

As described above, the SAI wavefront sensor may be used in a DICmicroscope system in accordance with one or more embodiments of theinvention. In one or more embodiments, the structured-aperturescomprises four holes (1 μm diameter, 1 μm center-to-center spacing, andthe two long axes are in the orthogonal x- and y-directionsrespectively) defined in a sliver film (100 nm thick) above a CMOS imagesensor (e.g., the image sensor offered by Micron MT9V403™) (FIG. 12(c)). The holes and the CMOS sensor are separated by an 80 μm thick(depicted as “d” in FIG. 12( b) (measured by a Thorlabs™ opticalcoherence tomography system [e.g., OCMP1300SS™]) layer of SU-8 resin. Byplacing the SAI wavefront sensor in the image plane of a standardmicroscope system, the four holes will selectively transmit and combinethe light fields from four adjacent points on the image to create aninterference pattern on the CMOS sensor. The total transmission of theinterference is proportional to the average image intensity at theaperture. In addition to the spacer thickness “d”, the offsetsoffset_(x)(x,y) and offset_(y)(x,y) of the zero-order interference spotare related to the net wavefront gradient G_(x)(x,y) and G_(y)(x,y) atthe aperture respectively [7]:

$\begin{matrix}{{{G_{x}\left( {x,y} \right)} = \frac{1}{\sqrt{1 + \left( \frac{d}{{offset}_{x}\left( {x,y} \right)} \right)^{2}}}}{{G_{y}\left( {x,y} \right)} = {\frac{1}{\sqrt{1 + \left( \frac{d}{{offset}_{y}\left( {x,y} \right)} \right)^{2}}}.}}} & (3)\end{matrix}$

The relative simplicity and absence of image intensity-related termsmakes this a particularly appealing way to measure the wavefrontgradient. In addition, this approach provides the ability to measure thewavefront gradient in both image-plane spatial dimensionssimultaneously.

The exact proportionality of a device may be experimentally determinedby measuring the interference pattern as illustrated in FIG. 12( d).Referring to FIG. 12( a), the SA wavefront sensor is illuminated with acollimated He—Ne laser beam (632.8 nm wavelength, 25 mm beam diameterand 4 mW power) from a range of incident angles. A least-square 2DGaussian fit is used to compute the total transmission and the offsets(offset_(x) and offset_(y)) of the zero-order spot in both x- andy-directions.

FIG. 12( b) shows the relationship between offset_(x)(offset_(y)) of thezero-order spot and the wavefront gradient G_(x)(G_(y)). Both curves areapproximately linear in the measurement ranges. This is consistent withthe geometric optics prediction: offset_(x)=d tan(θ)≈dG_(x), andsimilarly for offset_(y), where θ is the incident angle of the laserbeam and when the angle is small. The experimentally measuredproportionality from FIG. 12( b) was 70 μm while the predicted valuefrom thickness “d” measurement was 80 μm. Finally, it may be noted thatthis SA wavefront sensor works with broadband light sources and thezero-order interference spots coincide spatially for all wavelengths. Ascan be seen in FIG. 12( b), the offset of x of the zero-orderinterference spot of the structured aperture is linearly proportional tothe wavefront gradient Gx(Gy) along the x-(y-)direction in a definedmeasurement range.

FIG. 12( a) illustrates an experimental scheme of an SA-DIC microscopeused in accordance with one or more embodiments of the invention. Two20× objective lenses (Newport M-20×™) are aligned such that their rearconjugate planes (160 mm behind the objective lens) overlap. The sampleis placed at the front conjugate plane of the top objective (L1), andilluminated with a collimated white light (halogen lamp, 200 mW/cm²).Since the microscope system is symmetric, a 1:1 image of the sample isformed at the front conjugate plane of the bottom objective (L2). Thisimage equaled to convolution of the input sample light field with thePSF (point spread function) of the microscope system.

The SA wavefront sensor may be placed at the center of the image plane.Once placed, the sample may be raster-scanned (e.g., by two NewportCMH-25CCCL™ actuators) in the x-y plane to complete the mapping of theintensity and wavefront gradient of the image. During the imagingprocess, the illumination, the optical system and the sensor may befixed with respect to each other. Such positioning/fixation providesstable and precise measurement with a simple optical system.

To demonstrate the quantitative DIC sensing capability and itsapplication in biological imaging, a starfish embryo (CarolinaScientific™) may be used as a test sample. FIG. 13( a) shows amicroscope (e.g., Olympus BX41™) image of the sample acquired with a 10×objective. FIG. 13( b) is the corresponding image under a standard DICmicroscope (Zeiss Axioplan™, 40× objective). FIG. 13( c-e) show theintensity, X and Y DIC phase images acquired by a microscope with asingle scan in accordance with one or more embodiments of the invention.The spatial sampling step size was 0.5 μm in both x- and y-directions,and the exposure time associated with each sampling point was 8 ms.

The conventional transmission microscope image and the SA-DIC intensityimage are consistent with each other. However, the SA-DIC phase images(i.e., FIG. 13( c)-13(e) appear to be different from the conventionalDIC image. Such differences result because the SA-DIC phase imagespurely maps the wavefront gradients while the conventional DIC imagecontains some intensity image variations. This distinction isparticularly apparent when one compares the embryo's gastrocoel for allof the images. The image intensity associated with the gastrocoel regionwas low and the region appeared darker in the conventional DIC image. Incomparison, the corresponding areas of the SA-DIC phase images do notappear darker because they are pure phase maps. Finally, it may be notedthat the SA-DIC phase and amplitude images are also an improvement overconventional DIC images in that they are quantitative maps. Thewavefront gradient sensitivity of operating in the above describedexperimental conditions is approximately 4 mrad; the sensitivity can beimproved by using a better sensor platform, increasing measurement timeand/or increasing the illumination intensity. [7]

The ability to image birefringence samples properly is yet anotheradvantage of embodiments of the present invention. Birefringent objects,such as the potato starch storage granules, can alter the polarizationof the two displaced light fields in a conventional DIC microscope, suchthat the subsequent combination of the two fields is no longerdescribable by Eq. (1). This can give rise to Maltese-cross-like patternartifacts in the resulting conventional DIC images (as illustrated inFIG. 14( b). Since the SA-DIC microscope uses unpolarized illuminationand does not rely on polarization for image processing, it can imagebirefringent samples without artifacts, as shown in FIGS. 14( d) and(e). It is also worth noting that the dark absorption spots of thestarch granules in the center of the intensity images (FIG. 14( c)) donot appear in SA-DIC phase images (FIGS. 14( d) and (e)). This isanother clear indication that the SAI-DIC microscope can separate theintensity variations of the image wavefront from the phase variations.

FIG. 15 illustrates a completed wavefront-sensing sensor chip (topleft), a camera port housing for the chip (top right), and the use ofthe entire unit plugged into a camera port of a microscope in accordancewith one or more embodiments of the invention. Such a use provides forthe integration of the DIC functionality of the microscope onto thedetector whereas conventional DIC microscopy methods use bulky opticalelements in the microscope itself.

The aperture size may impact several aspects of the system performance.The SA-DIC microscope resolution may be limited by the numericalaperture of the collection optics or the aperture size divided by thesystem's magnification (M). By limiting the system magnification, aresolution equal to the aperture size (2 μm) may result (e.g., animaging system with M=1). A system can be created that is limited by thenumerical aperture of the collection optics by either increasing M ordecreasing the aperture size.

A smaller aperture can lead to a decreased sensitivity of the wavefrontgradient measurement for two reasons. Firstly, a smaller aperture willlead to a broader interference pattern which can negatively impact theability to determine the offsets of the zero order intereference spot[8]. Secondly, a smaller aperture will transmit less light and thereforelead to an overall decease in the detected signal.

In conclusion, one can demonstrate a high-resolution and artifact-freequantitative DIC microscopy method based on the SA wavefront sensor. Themethod can simultaneously generate one intensity and two orthogonalwavefront gradient images. Unlike a conventional DIC microscope, SA-DICis capable of imaging birefringent samples without artifactual imageerrors. Further, imaging speed can be increased by using more sensitivedetectors, e.g. avalanche photodiode (APD) array, employing fasterscanning system, and/or parallelizing the imaging process [9][10].

Conclusion

As described above, one or more embodiments of the invention providevarious different types of and methods for utilizing a wavefront sensor.A wavefront microscope or camera may include a light source, a wavefrontrelay system, a wavefront sensor, and an output mechanism. DICfunctionality of the microscope or camera may be integrated into thewavefront sensor.

The light source of the microscope or camera is configured to illuminatea sample.

The wavefront relay system is configured to relay a wavefront generatedby the sample.

The wavefront sensor is configured to receive the wavefront from therelay system, measure a local intensity of the wavefront, and measure aphase gradient of the wavefront. The wavefront sensor may be a singlepixel wavefront sensor where the different measurements and image mapsare obtained via raster scanning. Alternatively, the sensor may be awavefront sensor array where the measurements and images consist of asnapshot of the entire sample.

The wavefront sensor may be configured as a Shack-Hartmann type devicethat consists of a microlenses array that focuses the wavefront acrosseach lens of the array and forms a spot onto a photosensor array. Thephotosensor array outputs a measurement of the phase gradient of thewavefront and the local intensity of the wavefront.

Whether used in the wavefront microscope or in any other type of imagingdevice, embodiments of the invention may provide a specific type ofwavefront sensor using structured apertures. In such a device, a metalfilm has one or more patterned structured 2D apertures configured toconvert a phase gradient of the wavefront into a measurable form. Aphotodetector array receives the wavefront projected through theapertures and outputs a measurement of the phase gradient and localintensity of the wavefront.

The output mechanism outputs a local intensity 2D image map and a phasegradient 2D image map (e.g., onto a display device). In addition, theoutput mechanism may further analyze the data. Such an analysis mayreceive a recording of signals of the wavefront projected onto thephotosensor array. A computer (e.g., computer processor) may be used toseparate the signals from the projection of one patterned structuredaperture from the other apertures. Such a separation may be performed bysuppressing the crosstalk from neighbor hole projections iteratively.All signals of the projections for each aperture are summed together andis proportional to an intensity of the wavefront. A center of theprojection from each aperture is predicted. A center position changefrom the predicted center (based on snapshots taken with and without thesample in place) is determined and is directly related to the phasegradient of the wavefront (i.e., of the sample).

The foregoing description of the preferred embodiment of the inventionhas been presented for the purposes of illustration and description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed. Many modifications and variations are possiblein light of the above teaching. It is intended that the scope of theinvention be limited not by this detailed description, but rather by theclaims appended hereto.

REFERENCES

[1] C. J. Cogswell and C. J. R. Sheppard, Journal of Microscopy-Oxford165, 81 (1992).

[2] M. W. Davidson, M. Abramowitz, “Differential Interference Contrast,“Comparison of Wavefront and DIC Microscopy”, Florida State Universitywebsite (1998-2005).

[3] Ben C. Platt and Roland Shack, History and Principles ofShack-Hartmann Wavefront Sensing, Journal of Refractive Surgery 17,S573-S577 (September/October 2001).

[4] Shack-Hartmann, Wikipedia (Mar. 30, 2009).

[5] M. J. Booth, M. A. A. Neil, R. Juskaitis, T. Wilson, Proceedings ofthe National Academy of Sciences of the United States of America 99,5788 (April 2002).

[6] J. Z. Liang, D. R. Williams, D. T. Miller, Journal of the OpticalSociety of America a-Optics Image Science and Vision 14, 2884 (November1997).

[7] M. Lew, X. Cui, X. Heng and C. Yang, Optics Letters 32, 3 and 2963(2007).

[8] R. E. Thompson, D. R. Larson and W. W. Webb, Biophysical Journal 82,2775 (2002).

[9] X. Heng, D. Erickson, L. R. Baugh, Z. Yaqoob, P. W. Sternberg, D.Psaltis and C. H. Yang, Lab on a Chip 6, 1274 (2006).

[10] X. Cui, X. Heng, J. Wu, Z. Yaqoob, A. Scherer, D. Psaltis and C.Yang, Optics Letters 31, 3161 (2006).

[11] P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T.Colomb and C. Depeursinge, Optics Letters 30, 468 (2005).

[12] W. Choi, C. Fang-Yen, K. Badizadegan, S. Oh, N. Lue, R. R. Dasariand M. S. Feld, Nature Methods 4, 717 (2007).

[13] Xiquan Cui, Matthew Lew, and Changhuei Yang; “Quantitativedifferential interference contrast microscopy based onstructured-aperture interference”, Applied Physics Letters Vol 93 (9),091113 (2008); NIHMSID: 111214)

[14] John Daniel Kraus and Daniel A. Fleisch, Electromagnetics withApplications (5^(th) Ed)., Chapters 4-16 (1999).

1. A wavefront microscope comprising: (a) a light source configured toilluminate a sample; (b) a wavefront relay system configured to relay awavefront generated by the sample; (c) a wavefront sensor configured to:(i) receive the wavefront from the wavefront relay system; (ii) measurea local intensity of the wavefront; and (iii) measure a phase gradientof the wavefront; and (d) an output mechanism for outputting a localintensity two dimensional (2D) image map and a phase gradient 2D imagemap.
 2. The wavefront microscope of claim 1, wherein (a) the wavefrontsensor comprises a single pixel wavefront sensor; and (b) the localintensity 2D image map and phase gradient 2D image map are obtained viaraster scanning.
 3. The wavefront microscope of claim 1, wherein: (a)the wavefront sensor comprises a wavefront sensor array; and (b) thelocal intensity 2D image map and phase gradient 2D image map comprise asnapshot of the entire sample.
 4. The wavefront microscope of claim 1,wherein the wavefront sensor comprises: (a) a metal film having one ormore patterned structured 2D apertures configured to convert a phasegradient of the wavefront into a measurable form; (b) a photodetectorarray configured to: (i) receive the wavefront projected through the oneor more 2D apertures; and (ii) output a measurement of: (1) the phasegradient of the wavefront; and (2) the local intensity of the wavefront.5. The wavefront microscope of claim 1, wherein the wavefront sensorcomprises: (a) a microlenses array configured to focus the wavefrontacross each lens of the microlenses array and form a spot onto aphotosensor array; (b) the photosensor array configured to output ameasurement of: (i) the phase gradient of the wavefront; and (ii) thelocal intensity of the wavefront.
 6. The wavefront microscope of claim1, wherein differential interference contrast (DIC) functionality of thewavefront microscope is integrated into the wavefront sensor.
 7. Anwavefront sensor comprising: (a) a metal film having one or morepatterned structured two dimensional (2D) apertures configured toconvert a phase gradient of a wavefront into a measurable form; (b) aphotodetector array configured to: (i) receive the wavefront projectedthrough the one or more 2D apertures; and (ii) output a measurement of:(1) a phase gradient of the wavefront; and (2) an intensity of thewavefront.
 8. The wavefront sensor of claim 7, wherein multipleappertures are arranged into a two-dimensional wavefront sensor array.9. The wavefront sensor of claim 7, wherein the patterned structured 2Dapertures comprise a Fresnel zone plate.
 10. A computer-implementedmethod for analyzing data in a structured aperture interference (SAI)differential interference contrast (DIC) sensor comprising: (a)receiving, in a computer, from a photosensor array, a recording ofsignals representative of a wavefront projected onto the photosensorarray, wherein: (i) the wavefront is projected via one or more patternedstructured two-dimensional (2D) apertures in a metal film; and (ii) thesignals are recorded from the projection through every patternedstructure aperture; (b) separating, in the computer, the signalsrecorded from the projection of one patterned structure aperture fromother pattern structured apertures; (c) predicting, in the computer, acenter of the projection of each patterned structure aperture; (d)summing, in the computer, all signals of the projections for eachpatterned structure aperture, wherein the sum is proportional to anintensity of the wavefront; (e) determining, in the computer, a centerposition change from the predicted center of the projection, wherein thecenter position change is directly related to a phase gradient of thewavefront; and (f) outputting image data for the intensity and phasegradient.
 11. The computer-implemented method of claim 10, wherein theseparating comprises suppressing crosstalk from neighbor holeprojections iteratively.
 12. The computer-implemented method of claim10, wherein the determining of the center position change comprises acomparison to a predicted center based on a sample generated-wavefrontand a wavefront without a sample.
 13. A method for measuring a wavefrontin a wavefront microscope comprising: (a) illuminating a sample; (b)relaying a wavefront generated by the sample; (c) receiving the relayedwavefront at a wavefront sensor; (d) measuring, using the wavefrontsensor, a local intensity of the wavefront; (e) measuring, using thewavefront sensor, a phase gradient of the wavefront; and (f) outputtinga local intensity two dimensional (2D) image map and a phase gradient 2Dimage map based on the local intensity and phase gradient.
 14. Themethod of claim 13, wherein (a) the wavefront sensor comprises a singlepixel wavefront sensor; and (b) the local intensity 2D image map andphase gradient 2D image map are obtained via raster scanning.
 15. Themethod of claim 13, wherein (a) the wavefront sensor comprises awavefront sensor array; and (b) the local intensity 2D image map andphase gradient 2D image map comprise a snapshot of the entire sample.16. The method of claim 13, wherein the wavefront sensor comprises: (a)a metal film having one or more patterned structured 2D aperturesconfigured to convert a phase gradient of the wavefront into ameasurable form; (b) a photodetector array configured to: (i) receivethe wavefront projected through the one or more 2D apertures; and (ii)output a measurement of: (1) the phase gradient of the wavefront; and(2) the local intensity of the wavefront.
 17. The method of claim 13,wherein the wavefront sensor comprises: (a) a microlenses arrayconfigured to focus the wavefront across each lens of the microlensesarray and form a spot onto a photosensor array; (b) the photosensorarray configured to output a measurement of: (i) the phase gradient ofthe wavefront; and (ii) the local intensity of the wavefront.
 18. Themethod of claim 13, wherein differential interference contrast (DIC)functionality of the wavefront microscope is integrated into thewavefront sensor.
 19. A method for detecting a wavefront comprising: (a)projecting a wavefront through a metal film having one or more patternedstructured two dimensional (2D) apertures onto a photodetector array;(b) receiving, in the photodetector array, the wavefront projectedthrough the one or more 2D apertures; and (c) outputting a measurementof: (1) a phase gradient of the wavefront; and (2) an intensity of thewavefront.
 20. The method of claim 19, further comprising arrangingmultiple appertures into a 2D wavefront sensor array.
 21. A method formeasuring a wavefront in a wavefront camera system comprising: (a)illuminating an object; (b) relaying a wavefront generated by theobject; (c) receiving the relayed wavefront at a wavefront sensor; (d)measuring, using the wavefront sensor, a local intensity of thewavefront; (e) measuring, using the wavefront sensor, a phase gradientof the wavefront; and (f) outputting a local intensity two dimensional(2D) image map and a phase gradient 2D image map based on the localintensity and phase gradient.